Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator, signals via the S1P receptors (S1PnR) to regulate the development, homeostasis and phenotypic changes of the vascular system. Our recent studies have shown that the Gi-, Rac- and Akt-coupled S1P1R is induced in angiogenic vessels, is required for tumor angiogenesis, orchestrates cell-cell adhesion molecule function, promotes vascular maturation/stabilization and inhibits vascular permeability. In contrast, we found that S1P2R, which activates the Rho GTPase- dependent activation of the tumor suppressor phosphatase PTEN, inhibits endothelial cell migration, disrupts cell-cell junctions, induces vascular permeability, inflammatory gene expression and pathological angiogenesis. Given that S1P is abundant in plasma, spatially- and temporally-regulated expression of S1P receptors in endothelial cells is a major determinant of the biological response of S1P in a given vascular bed. The long-term goal of this research program is to better understand the logic of S1P signaling in the vasculature. Since the first generation S1P receptor modulator called FTY720 is currently undergoing clinical trials (phase III) for multiple sclerosis and transplant rejection, the findings from this research program may provide critical information that is directly translatable to the clinic. The central hypothesis of this proposal is that S1P profoundly regulates the phenotype of vascular endothelium by temporally- and spatially-precise signaling of S1P1R and S1P2R. In the first specific aim, we will test the hypothesis that S1P1R is essential for physiological angiogenesis. The ability of S1P1R-regulated signaling pathways to promote pericyte recruitment, and inhibit vascular permeability, and inflammation will be investigated using genetic mouse models and pharmacological tools. Secondly, we will address the question of how the S1P receptor signaling in the endothelium is attenuated given the fact that the ligand is highly abundant in plasma. We will critically test the hypothesis that selective subcellular targeting of S1P1R, namely, recycling between endosomes and plasma membrane or ubiquitin-based targeting to degradative organelle is an important aspect of regulation and decipher the molecular mechanisms involved. Thirdly, we will define the significance of S1P2R signaling in pathological angiogenesis. We will test if S1PR2 coupling to Rho/ROCK/PTEN and p38 pathways induces vascular permeability and inflammatory gene expression, respectively. Molecular mechanisms of receptor coupling, regulation of endothelial junctional integrity and transcriptional regulation will be addressed. And finally, we propose to define the coordinate signaling of S1P1R and S1P2R in vascular homeostasis and development. A hypermorphic S1P1R that does not internalize will be knocked into the mouse genome and the resultant tissue angiogenesis, vascular stabilization, vascular permeability and inflammation will be characterized. The ability of S1P2R to modulate S1P1R-dependent effects will be tested by varying the gene dosage of S1p2r in the context of the S1P1R hypermorph. These studies are anticipated to enhance our understanding of S1P function in the vasculature. This information may be useful in the control of vascular inflammation and angiogenesis by S1P receptor modulators. Narrative This proposed research will investigate the role of sphingosine 1-phosphate receptors 1 and 2 (S1P1R and S1P2R) on vascular endothelial cell biology and pathophysiology. The concept that S1P1R and S1P2R mediate opposing actions to regulate physiological and pathological angiogenesis will be examined in detail using in vitro cell culture systems to investigate the mechanisms involved. Mouse models of angiogenesis in the retina will be used to obtain in vivo correlates. Finally coordinate signal transduction of these two receptors will be examined. A novel mouse model that will allow us to examine the coordinate signaling will be developed using an internalization deficient mutant of S1P1R. These studies are expected to derive critical insights into the workings of S1P receptors, which are important drug targets to control the vascular and immune systems.